The overall goal of this research is to understand the mechanisms for depressed crossbridge cycling in the intact ventricle in chronic overload states in particular during the transition from compensatory hypertrophy to cardiac failure. Depressed crossbridge cycling caused by reduced myofibrillar ATPase activity is a key abnormality in overloaded myocardium. To investigate the mechanisms underlying the transition from compensatory left ventricular hypertrophy to failure, the relationship between LV mechanoenergetics and contractile proteins in experimental models of cardiac hypertrophy with transition to heart failure will be studied. The basis for this hypothesis is from preliminary data in DSS rats, where the transition from compensatory LV hypertrophy to cardiac failure is associated with depressed Emax and increased contractile efficiency and economy. These changes, which are closely linked to myofibrillar ATPase activity, cannot be explained solely on the basis of isomyosin switching (V1 to V3) but are temporally correlated with altered expression of troponin T isoforms. The following hypotheses are suggested: (1) Contractile efficiency and economy are tightly correlated with depressed crossbridge cycling as assessed in skinned strips, (2) a component of depressed ATPase activity is caused by altered thin filament regulation as assessed by in vitro motility assay, and (3) the molecular mechanism that accounts for depressed crossbridge cycling are alterations in thin filament isoform expression and contractile protein phosphorylation. Ventricular mechanoenergetics expressed as the inverse slope of VO2-PVA relation and inverse slope of VO2-forced time interval relation in isolated perfused hearts will be measured and correlated to alterations in crossbridge kinetics (assessed by actomyosin ATPase rate and frequency of maximal oscillatory work (Fmax) in skinned myocardial strips) and correlated to changes in velocity, ATPase rate and force production of myosin interacting with native thin filaments (in vitro motility assay). Changes in TnT protein isoform switching and phosphorylation in addition to TnI and Tm protein levels will be measured by immunoblotting and 2D gel electrophoresis in the presence and absence of alkaline phosphatase.